1. Field of the Invention
The invention described herein provides a non-toxic method of increasing the natural mutation rate in bacteria. The method relies on the use of a modified salt namely thio-phosphate as a source of phosphate in culture media. Thio-phosphate is taken up by cells and ultimately incorporated into DNA such that it inhibits DNA editing and other cellular repair pathways. The mutation rate can be increased several hundred fold. Thio-phosphate substituted media is useful for generating variant strains, plasmids, or phage DNAs with high efficiency after several cycles of growth and amplification in thio-phosphate modified media.
2. Description of Related Disclosures
Traditional methods of mutagenesis involve chemical mutagens such as ethylmethane sulfonate (EMS) and others that are toxic carcinogens (Lawrence (1991) Methods and Enzymology 194:273-281). Such mutagens can be used to generate base substitutions, deletions, frame shift mutations, and additions. Another method of mutagenesis involves the use of transposable elements that when activated transpose and insert in or near a gene resulting in altered gene expression (Spradling (1999) Genetics 153:135; Rothstein (1991) Methods and Enzymology 194:281-301; Spradling and Rubin (1982) Science 218:341-347).
The mutagenesis of specific gene segments is an important tool in the field of biotechnology. Early work involved the chemical mutagenesis of recombinant DNA plasmids in vitro followed by transformation of cells in vivo (Sikorski and Boeke (1991) 194:302-318). More recent methods involve the use of mutator strains for growing plasmids (Greener et al (1996) Methods Mol. Biol. 57: 375-385) or in vitro mutagenesis of plasmids via error prone PCR (Cline et al (1996) NAR 24:3546-3551; Leung et al (1989) Technique 1; 11-15). A drawback of PCR mutagenesis has been a nonuniform mutational spectrum and low yields resulting from the required reaction conditions. Efforts to overcome such biases have been devised through the use of new enzymes (Cline and Hogrefe (2000) Strategies 13:157-161). Expanding the power of gene specific mutagenesis is the technique of PCR shuffling that is used to generate new combinations of mutant alleles for specific genes (Stemmer (1994) PNAS 91:10747-10751). The method involves the introduction of new mutations which serve as a source of genetic diversity for subsequent recombination events.
The present invention describes methods for the use of thio-phosphate as a mutagenic agent. Previous work has shown that phosphorothioate substituted DNA is resistant to DNA editing by DNA polymerase in vitro (Burgers and Eckstein (1979) J. Biol. Chem. 254:68896893). More recently, it has been shown (Frayne U.S. Ser. No. 10/007,489, Filed Dec. 5, 2001) that thio-phosphate can replace normal phosphate in cells and result in the incorporation of thio-phosphate nucleotides into DNA creating phosphorothioate linkages. Thus thio-phosphate can be used in culture media to increase the natural mutation rate by inhibiting DNA repair mechanisms in vivo. Cells can be grown for extended periods in media with thio-phosphate fully substituting for phosphate. The 100-200 fold increase in mutation rate is greater than the 20 fold increase expected from in vitro studies (Goodman et al (1993) Crit. Rev. Biochem. Mol. Biol. 28:83-126). Multiple repair pathways are presumably blocked by phosphorothioate linkages in bacteria. In contrast to bacteria yeast appear to show little or no increase in mutation rate when thio-phosphate is used as a source of phosphate in culture media. The enhanced mutation rate provides a novel means to mutate cells or recombinant DNA sequences in vivo and also allows for a wide range of mutations.